The primary goal of the proposed work is to define the mechanisms responsible for specificity of function within the E2F family of transcription factors. The importance of the work lies in the fact that this group of transcription factors, that are targets for control by the Rb tumor suppressor, have been shown to be critical for cell proliferation, including the transition through the G1/S phase and G2/M of the cell cycle. In addition, E2Fs also link the proliferative response with cell fate decisions including apoptosis. Various studies suggest that these various roles are mediated by individual members of the E2F family, at least in part through the control of different sets of target genes. Our recent work points to a mechanism for promoter specificity that involves the coordinated action of E2F proteins with partner transcription factors whereby specific combinations of factors are required for proper promoter function. This concept of combinatorial control has been put forward as a model for achieving the massive complexity of transcription control for the large number of protein-coding genes using a limited number of transcription factors. We now propose to extend these initial studies to more globally define the role of E2F protein interactions in the determination of functional specificity. This will include the use of genome-scale data analysis to predict E2F interactions that impart specificity of transcription control that will then be verified with specific assays of promoter interaction and function. In addition, we will identify structure features of E2Fs and cooperating transcription factors that are essential for this combined action and we will finally explore mechanism that control gene expression through a control of these protein interactions. Given the pervasive role of Rb pathway mutations in human cancer, understanding the specificity of function in the pathway, particularly the action of the E2F proteins, is critical in considering the development of cancer therapeutics that might target the most relevant aspects of Rb function. PUBLIC HEALTH RELEVANCE: The E2F family of transcription factors are now recognized as central to the control of cellular proliferation. Loss of E2F control through deregulation of the Rb pathway is also observed as a common if not mandatory consequence of oncogenesis. As such, the understanding of the mechanisms by which these proteins control gene expression is clearly critical to understanding aspects of cell proliferation control and the contribution to human cancer.